With advances in mass spectrometric technologies and bioinformatics, it is possible to identify the majority of proteins from a cell extract after separation by two-dimensional electrophoresis (2-DE). However, amassing a catalog of proteins within a cell (the proteome) is of limited practical use without some knowledge of the functional state of those proteins. Reversible protein phosphorylation is the most abundant post-translational modification in eukaryotes and as such plays a crucial role in regulating protein function in both normal homeostatic processes as well as disease processes. Thus, identification and characterization of the phosphoproteins present within a cell under various conditions (the phosphoproteome) will generate a more useful molecular census of normal versus abnormal cellular states.
Given its crucial role in biology, the study of protein phosphorylation as it relates to normal and pathological physiology is one of the most important avenues of contemporary biomedical research. Specifically, identification of the major kinase and phosphatase substrates that are relevant to a given cellular process is of paramount importance if, at a biochemical level, complex biological events such as cell division, differentiation, and movement are to be defined.
Although simply stated, this task is truly formidable. It is currently thought that nearly one-third of all cellular proteins contain, at one time or another, covalently bound phosphate. See e.g., Hunter, 2000; Cohen, 2000. In addition, genomic studies suggest that the human genome encodes more than 1000 protein kinases and nearly 500 protein phosphatases to effect these modifications. See Cohen, 2000. As such, substantial interest and effort is currently being placed in phosphoproteomics: the identification and characterization of catalogs of phosphoproteins, and the changes in their phosphorylation status under various physiological conditions. See e.g., Ahn & Resing, 2001; Conrads et al., 2002; Mann et al., 2002. Of particular interest in phosphoproteomics is the identification of specific differences between the phosphoproteomes of normal and diseased cells in order to extrapolate these differences into exploitable targets for pharmacologic therapies.
The success of phosphoproteomics depends on the ability to detect and subsequently identify phosphorylated proteins in complex mixtures isolated from various experimental sources. This, in turn, will require reliable detection methods that are compatible with contemporary protein identification technologies such as peptide microsequencing and mass spectrometry. The panel of currently available detection methods, however, leaves an urgent need for novel, more powerful methods.
Current methods for detecting or selectively enriching for phosphorylated proteins can be divided into three categories—radioisotopic, immunological, and chromatographic. While each method has been used successfully under certain conditions, each suffers from its own significant limitations as well. See e.g., Kaufmann et al., 2001; Conrads et al., 2002.
Using radioisotopic methods, phosphoproteins are most commonly detected by autoradiography of individual proteins or protein mixtures isolated from cells cultured in the presence of phosphate containing 32P (or, less frequently, 33P) at its core. While the use of radioactive phosphate provides a fairly sensitive method to label a large number of proteins using a technically uncomplicated procedure, it has several significant drawbacks. Radioactive labeling requires “metabolic access” to the biological sample (e.g. cultured cells), precluding its application to tissues and clinical samples. Additionally, even when access is available, the labeling period itself poses problems. It must be long enough to allow free radioactive phosphate to equilibrate with the cellular ATP pool, but brief enough to prevent cellular damage or stress due to the radioisotope or the reduced concentration of total phosphate in typical labeling media. Also, differences in phosphate turnover among proteins and individual amino acids result in preferential labeling of proteins with rapid phosphate turnover rates as well as unequal incorporation of 32P into serine vs. threonine vs. tyrosine residues. Sefton, 1991. Furthermore, the use of radioactivity presents considerable hazardous material concerns and waste storage/disposal costs. The final, and arguably most significant, limitation of radioactive phosphate labeling is that the radioactive samples generated are almost universally rejected by commercial protein sequencing and mass spectrometry (MS) facilities. Thus, the precious sample(s) must be either enzymatically dephosphorylated (which can be technically difficult and prevents subsequent determination of the modified residue(s)) or kept shelved and unanalyzed for weeks or months until the level of radioactivity has decayed to background levels.
Some of the shortcomings of radioisotopic labeling can be addressed by using antibodies that specifically recognize the phosphorylated forms of certain amino acids. Principally, antibody-based methods do not require prior labeling or other manipulation of the biological source of phosphoproteins and they present no real safety or hazardous material concerns. The relevant antibodies fall into two classes. Phosphoamino acid antibodies (PAAAs) recognize the individual phosphorylated residue (i.e. p-Ser, p-Thr, or p-Tyr) regardless of the surrounding amino acid composition. Phospho-sequence specific antibodies (PSSAs) recognize the phosphorylated residue only within the context of a specific amino acid sequence. While PSSAs have proven useful in the analysis of single, specific phosphoproteins (e.g. mitogen-activated protein kinase, retinoblastoma protein), their specificity precludes their use in identifying unknown phosphoproteins in complex mixtures.
PAAAs are more useful in general phosphoproteomic analyses, but still suffer limitations. Antibodies against p-Tyr have proven most useful. See e.g., Cooper et al., 1983; Kaufmann et al, 2001. Generally, they are of sufficiently high affinity to allow detection of low-abundance proteins by immunoblotting and can also be used to enrich tyrosine-phosphorylated proteins by immunoprecipitation and immunoaffinity chromatography. Their ability to specifically recognize p-Tyr regardless of the surrounding sequence is less than optimal, however. See e.g., Cooper et al., 1983; Kaufmann et al., 2001; Conrads et al, 2002; Mann et al., 2002.
The major limitation for p-Tyr antibodies in phosphoproteomics is, again, their specificity. While tyrosine-phosphorylated proteins are an important fraction of total cellular phosphoproteins, there are also the least abundant by far. Indeed, the ratio of p-Ser, p-Thr, and p-Tyr in cells is estimated to be approximately 1800:200:1. Mann et al., 2002. Thus, use of p-Tyr antibodies can be expected to detect, at best, 0.05% of all phosphoproteins in a cell. Unfortunately, the use of anti-p-Ser and anti-p-Thr antibodies to detect the remaining 99.95% has not been very successful. Due to their relatively small size, these phosphoamino acids are substantially less antigenic than p-Tyr. Attempts to create anti-p-Ser and anti-p-Thr antibodies have resulted in antibody preparations that are alternatively 1) specific but low-affinity; 2) high-affinity but bind to p-Ser, p-Thr, and p-Tyr with comparable strength; or 3) high-affinity but bind only a restricted subset of protein sequences containing their target phosphoamino acid. See e.g., Kaufmann et al., 2001; Mann et al., 2002. Thus, none of these antibodies are particularly useful to generalized phosphoproteomic analysis.
Other major complications of using immunological reagents for phosphoprotein detection are encountered after the actual detection. Immunoblotting is best performed after blocking unoccupied sites on the solid-phase support with protein solutions, which interferes with subsequent microchemical techniques required for protein identification. Indeed, the antibodies themselves can complicate identification of the target phosphoprotein. On the other hand, removal of the antibody requires relatively harsh treatments (e.g. heat plus detergent and reducing agents), which can negatively impact subsequent attempts at protein sequencing and mass spectrometry of the detected proteins. See e.g., Kaufmann et al., 2001; Conrads et al., 2002. Thus, while immunological techniques offer certain advantages over metabolic labeling, difficulties remain in applying current anti-phosphoamino acid antibodies as phosphoproteomic reagents.
A third method involves chromatographic separation of phosphorylated from non-phosphorylated proteins. Chromatographic separation of phosphoproteins or phosphopeptides can reduce “proteomic noise” by eliminating non-phosphorylated proteins from analytical samples before MS. The use of phospho-immunoaffinity columns has been employed, but this strategy suffers from the limitations discussed above for other antibody-based methods.
Other techniques have been described that employ specific chemical derivatization of phosphate groups with heterologous functional groups that allow selective chromatographic separation of the once-phosphorylated species. Oda et al., 2001; Zhou et al., 2001. One such approach involves replacement of the phosphate groups of serine and threonine phosphopeptides by ethanedithiol via a β-elimination reaction, followed by tagging the derived peptides with a biotin affinity tag, which allows separation from non-phosphorylated peptides by avidin affinity chromatography. Oda et al., 2001. One drawback of this approach is the low reactivity of phosphothreonine and non-reactivity of phosphotyrosine residues in β-elimination reactions.
A second approach involves alkylation of existing cysteine residues followed by carbodiimide-catalyzed reaction of phosphates with cystamine to introduce a free sulfhydryl group, which allows derivatized peptides to be captured on iodoacetic beads. Zhou et al., 2001. While broadly reactive with all three major phosphoamino acids, the method involves a six-step derivatization/purification protocol that requires more than 13 hours to complete and produces only a 20% yield.
Thus, these techniques have their own significant and unique drawbacks. They also suffer from the same major limitation that all other chromatographic methods do, namely that a given method will (theoretically) retain any and all phosphoproteins in a given sample, even those for which the phosphorylation state does not change between relevant “before and after” conditions. This inability to cull experimentally relevant targets from the entire pool of cellular phosphoproteins introduces significant “noise” into the data, the elimination of which is a significant ongoing difficulty in the art. See e.g., Oda et al., 2001; Steen et al., 2001; Zhou et al., 2001; Ficarro et al., 2002; Mann et al., 2002.
Thus, there exists a long-felt and continuing need in the art for new methodologies that will allow rapid, safe, specific, and complete phosphoprotein detection that are compatible with mass spectrometric identification techniques. The presently claimed subject matter addresses this and other needs in the art.